Chlorine Dioxide Pilot Study

From 9-3-14.

Lawns of B. thuringiensis has been growing on LB only agar at 30C for three days. Of the three plates containing growth, one had its spores isolated using the ice-water washing method previously performed on P. larvae. 

Spore isolation method for B. thuringiensis:
1. Add 10 mL of sterile ddH2O to surface of agar plate
2.Gently rub the surface of the agar with a sterile cell spreader to dislodge attached bacteria
3. Carefully pipet up the 10 mL and add to 50 mL tube
4. Add an additional 5 mL of H2O to agar surface, rub, and add to 50 mL tube
5. Allocate the 15 mL volume into several 1.5 mL eppendorf tubes
6. Centrifuge tubes at 16,000 g for 1 minute
7. Carefully discard supernatant and upper pellet layer
8. Wash step: Re-suspend the pellet in 1 mL of sterile ddH2O and centrifuge again
9. Repeat wash step for a total of 5 times (consolidate to only one tube over the course of the washes)
10. Heat pellet in water bath at 65C for 30 minutes (use Gibbs lab water bath)
11. Repeat wash steps again for a total of 5 times.
12. After the last wash, re-suspend the pellet in 500 uL of sterile ddH2O
13. Store pellet stock at 4C

Note: incubated in water bath for 30 minutes for B. thuringiensis, only 15 for P. larvae

Below is an image of what the B .thuringiensis plate looked like before its spores were harvested:

The B. thuringiensis spore stock was diluted 1:10 down to 10^-4 in sterile ddH2O and the drop plate method was performed on LB only agar to quantify CFU/mL of the stock. Agar plate was incubated at 30C not inverted.

It should be noted that it was more difficult to remove the surface bacteria of B. thuringiensis using the cell scraper compared to removing the P. larvae on MYPGP agar. Also, the spore pellet the B. thuringiensis was not as visible as it was in the P. larvae spore preps after seven days. This could be that B. thuringiensis had not been allowed to incubate long enough to peak sporulation on LB.

The additional two LB only plates containing three day old lawns of B. thuringiensis are continued to incubate at 30C for additional days.


I received a number of packages from the USDA  ordered from VWR on Friday (9-5-14) that had been ordered for my chlorine dioxide study. Today I received an additional package from the USDA ordered from Sigma-Aldrich. Boxes are stored in Fisher lab and inventory of them will follow soon. They are pictured below:

//EWW